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Immune surveillance cells such as T cells and phagocytes utilize integral plasma membrane receptors to recognize surface signatures on triggered and activated cells such as those in apoptosis. One such family of plasma membrane sensors, the transmembrane immunoglobulin and mucin domain (Tim) proteins, specifically recognize phosphatidylserine (PS) but elicit distinct immunological responses. The molecular basis for the recognition of lipid signals on target cell surfaces is not well understood. Previous results suggest that basic side chains present at the membrane interface on the Tim proteins might facilitate association with additional anionic lipids including but not necessarily limited to PS. We, therefore, performed a comparative quantitative analysis of the binding of the murine Tim1, Tim3, and Tim4, to synthetic anionic phospholipid membranes under physiologically relevant conditions. X-ray reflectivity and vesicle binding studies were used to compare the water-soluble domain of Tim3 with results previously obtained for Tim1 and Tim4. Although a calcium link was essential for all three proteins, the three homologs differed in how they balance the hydrophobic and electrostatic interactions driving membrane association. The proteins also varied in their sensing of phospholipid chain unsaturation and showed different degrees of cooperativity in their dependence on bilayer PS concentration. Surprisingly, trace amounts of anionic phosphatidic acid greatly strengthened the bilayer association of Tim3 and Tim4, but not Tim1. A novel mathematical model provided values for the binding parameters and illuminated the complex interplay among ligands. In conclusion, our results provide a quantitative description of the contrasting selectivity used by three Tim proteins in the recognition of phospholipids presented on target cell surfaces. This paradigm is generally applicable to the analysis of the binding of peripheral proteins to target membranes through the heterotropic cooperative interactions of multiple ligands., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Adding cholesterol to monolayers of certain phospholipids drives the separation of liquid-ordered from liquid-disordered domains. The ordered phases appear to contain stoichiometric complexes of cholesterol and phospholipid. Furthermore, it has been suggested that the cholesterol in these complexes has a low chemical activity compared to that of the free sterol; i.e., that in excess of the phospholipid binding capacity. We have now tested the hypothesis that the membrane intercalator 1-hexadecanol (HD) similarly associates with phospholipids and thereby displaces the complexed cholesterol. HD introduced into monolayers of pure dimyristoylphosphatidylcholine generated highly condensed (stable and solid) domains. In contrast, the phase behavior of mixed monolayers of the phospholipid, sterol, and alcohol suggested that HD could substitute for cholesterol mole for mole in promoting liquid-ordered domains. We also found that the transfer of cholesterol from mixed monolayers to aqueous cyclodextrin was greatly stimulated by the presence of HD, but only at levels sufficient to competitively displace the sterol from the phospholipid. This enhanced efflux was interpreted to reflect an increase in uncomplexed cholesterol. We conclude that HD forms complexes with dimyristoylphosphatidylcholine that are surprisingly similar to those of cholesterol. HD competitively displaces cholesterol from the phospholipid and thereby increases its chemical activity.

We probed the kinetics with which cholesterol moves across the human red cell bilayer and exits the membrane using methyl-beta-cyclodextrin as an acceptor. The fractional rate of cholesterol transfer (% s(-1)) was unprecedented, the half-time at 37 degrees C being ~1 s. The kinetics observed under typical conditions were independent of donor concentration and directly proportional to acceptor concentration. The rate of exit of membrane cholesterol fell hyperbolically to zero with increasing dilution. The energy of activation for cholesterol transfer was the same at high and low dilution; namely, 27-28 Kcal/mol. This behavior is not consistent with an exit pathway involving desorption followed by aqueous diffusion to acceptors nor with a simple one-step collision mechanism. Rather, it is that predicted for an activation-collision mechanism in which the reversible partial projection of cholesterol molecules out of the bilayer precedes their collisional capture by cyclodextrin. Because the entire membrane pool was transferred in a single first-order process under all conditions, we infer that the transbilayer diffusion (flip-flop) of cholesterol must have proceeded faster than its exit, i.e., with a half-time of <1 s at 37 degrees C.

Cells keep their cholesterol in balance by sensing its level in the endoplasmic reticulum and transducing this information into the expression of multiple homeostatic genes. Two recent papers from the Brown and Goldstein laboratory provide important new insights into how an integral ER protein, SCAP, mediates this process.

The role of the membrane skeleton in determining the shape of the human red cell was probed by weakening it in situ with urea, a membrane-permeable perturbant of spectrin. Urea by itself did not alter the biconcave disk shape of the red cell; however, above threshold conditions (1.5 M, 37 degrees C, 10 min), it caused an 18% reduction in the membrane elastic shear modulus. It also potentiated the spiculation of cells by lysophosphatidylcholine. These findings suggest that the contour of the resting cell is not normally dependent on the elasticity of or tension in the membrane skeleton. Rather, the elasticity of the skeleton stabilizes membranes against deformation. Urea treatment also caused the projections induced both by micropipette aspiration and by lysophosphatidylcholine to become irreversible. Furthermore, urea converted the axisymmetric conical spicules induced by lysophosphatidylcholine into irregular, curved and knobby spicules; i.e., echinocytosis became acanthocytosis. Unlike controls, the ghosts and membrane skeletons obtained from urea-generated acanthocytes were imprinted with spicules. These data suggest that perturbing interprotein associations with urea in situ allowed the skeleton to evolve plastically to accommodate the contours imposed upon it by the overlying membrane.

The molecular basis for the elasticity of the human erythrocyte membrane was explored. Skeletons were released from ghosts in Triton X-100 and their dimensions followed by dark-field microscopy and packed volume. The rest size of skeletons was assumed to reflect the balance point between expansion (deformation) driven by electrostatic repulsions among the excess of fixed negative charges on the proteins and contraction (recovery) driven by their elasticity. The size of skeletons decreased with increasing temperature. This finding suggests that entropy drives elasticity. The requisite entropy change could be associated with either the configurational freedom of flexible protein chains or with the solvation of side chains exposed during protein dissociation (hydrophobic effects). To distinguish between these two alternatives, we tested the impact of two weak denaturants, 10% ethanol and 20 nM lithium 3,5-diiodosalicylate. Both agents reversibly promoted the expansion of skeletons, presumably by reducing their elasticity. Since the conformation of random coils and globular proteins should not be significantly altered by these mild treatments, this finding strongly suggests a role for weak interdomain and/or interprotein associations. We conclude that the elasticity of the red cell membrane skeleton may not derive from the configurational entropy of flexible coils. Rather, the elastic energy may arise from reversible dissociations of weak but specific intramolecular and/or intermolecular contacts, presumably within deformed spectrin filaments.